JP2024525742A - KCNV2 gene therapy - Google Patents

Abstract

Provided herein are expression constructs, viral genomes, and vectors for expression of Kv8.2, as well as pharmaceutical compositions comprising the vectors disclosed herein. Also provided are methods of using the expression constructs and vectors disclosed herein, including methods of treating a retinal disease in a subject in need thereof, the retinal disease being associated with one or more mutations in the KCNV2 gene, comprising administering to the subject a vector disclosed herein.Selected Figure:

Description

The present disclosure relates generally to the fields of molecular biology and medicine. More specifically, the present invention provides compositions and methods for gene therapy for the treatment of retinal diseases.

Kv8.2 is a voltage-gated potassium channel subunit encoded by the KCNV2 gene. The KCNV2 gene is located on chromosome 9p24.2 and consists of two exons that code for a 545 amino acid protein. This protein is expressed in the inner segments (ellipsoid and myoid regions) of retinal rod and cone photoreceptors and is absent in the outer segments of humans, mice, and macaques. Kv8.2 interacts with other potassium subunits, such as Kv2.1, which is expressed in rod and cone inner segments, and Kv2.2, which is expressed in human cones but not rods. Kv8.2 further interacts with Kv2 channels to alter their biophysical properties.

Kv8.2 is the only potassium channel subunit previously implicated in human disease. Kv8.2 variants/mutations cause a severe inherited photoreceptor dystrophy known as "cone dystrophy with supernormal rod response" (CDSSR). Symptoms of CDSSR include reduced visual acuity, color vision abnormalities, and altered electroretinogram responses such as elevated b-wave amplitude.

Therefore, there is an urgent need for novel therapeutic approaches for the treatment of retinal diseases associated with KCNV2 mutations (including, but not limited to, CDSSR).

In one aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising:
An expression construct comprising: (a) a promoter sequence that confers expression in a photoreceptor cell; and (b) a nucleic acid sequence encoding Kv8.2;
The nucleic acid sequence is operably linked to a promoter to provide an expression construct.

In embodiments, the promoter sequence is a CAG or rhodopsin kinase (RK) promoter sequence. In embodiments, the promoter sequence comprises a sequence that is at least 90% identical to SEQ ID NO:8. In embodiments, the promoter sequence comprises the sequence of SEQ ID NO:8. In other embodiments, the promoter sequence comprises a sequence that is at least 90% identical to SEQ ID NO:7. In embodiments, the promoter sequence comprises the sequence of SEQ ID NO:7.

In embodiments, the expression construct further comprises a post-transcriptional regulatory element. In embodiments, the expression construct further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In embodiments, the WPRE comprises a sequence that is at least 90% identical to SEQ ID NO:11 or comprises the sequence of SEQ ID NO:11.

In an embodiment, the nucleic acid sequence encoding Kv8.2 is a coding sequence (cds) from the WT KCNV2 gene. In an embodiment, the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 90% identical to SEQ ID NO:9. In an embodiment, the nucleic acid sequence encoding Kv8.2 comprises a sequence that includes SEQ ID NO:9.

In an embodiment, the nucleic acid sequence encoding Kv8.2 is a codon-optimized KCNV2 gene sequence. In an embodiment, the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 90% identical to SEQ ID NO: 10. In an embodiment, the nucleic acid sequence encoding Kv8.2 comprises a sequence that includes SEQ ID NO: 10.

In some embodiments, the nucleic acid sequence encoding Kv8.2 encodes a protein comprising a sequence that is at least 90% identical to SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding Kv8.2 encodes a protein comprising SEQ ID NO: 13.

In an embodiment, the expression construct further comprises a bovine growth hormone polyadenylation (BGH-polyA) signal. In an embodiment, the polyadenylation signal comprises a sequence that is at least 90% identical to SEQ ID NO: 12. In an embodiment, the polyadenylation signal comprises SEQ ID NO: 12.

In an embodiment, the expression construct comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-4. In an embodiment, the expression construct comprises a sequence selected from the group consisting of SEQ ID NOs: 1-4.

In another aspect, the disclosure provides a vector comprising an expression construct disclosed herein. In an embodiment, the vector is a viral vector. In an embodiment, the vector is an adeno-associated viral (AAV) vector. In an embodiment, the vector comprises a genome derived from AAV serotype AAV2. In an embodiment, the vector comprises a capsid derived from AAV7m8. In an embodiment, the vector comprises a capsid derived from AAV5.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a vector disclosed herein and a pharma- ceutically acceptable carrier.

In another aspect, the disclosure provides a method for treating a retinal disease in a subject in need thereof, the retinal disease being associated with one or more mutations in the KCNV2 gene, comprising administering to the subject a vector or pharmaceutical composition disclosed herein. In an embodiment, the retinal disease is cone dystrophy with supernormal rod response (CDSSR).

In another aspect, the present disclosure provides a method for increasing expression of KCNV2 in a subject in need thereof, the method comprising administering to the subject a vector or pharmaceutical composition disclosed herein.

In another aspect, the present disclosure provides a method for increasing Kv8.2 levels in photoreceptors in a subject in need of increased Kv8.2 levels in photoreceptors, comprising administering to the subject a vector or pharmaceutical composition disclosed herein.

In embodiments, the vector or pharmaceutical composition is administered by intraocular injection. In embodiments of the disclosed methods, the vector or pharmaceutical composition is injected into the central retina of the subject.

Schematic diagrams of expression constructs: pCAG-KCNV2 WT, pCAG-KCNV2 Opti, pRT-KCNV2 WT, and pRT-KCNV2 Opti are shown. Shown are KCNV2 WT and KCNV2 Opti mRNA levels in HEK293 cells compared to KCNV2 WT and KCNV2 Opti mRNA levels in ARPE19 cells as determined by qPCR (48 hours after transfection). Kv8.2 immunofluorescence of ARPE19 cells transfected with pCAG-GFP (top row), pCAG-KCNV2 Opti (middle row), and pCAG-KCNV2 WT (bottom row) are shown. Scale bar = 10 μm. Data from HEK293 cells analyzed by FACS are shown. A shows FACS data showing mean fluorescence intensity (MFI) from three independent experiments for HEK293 cells transfected with pCAG-KCNV2 WT and pCAG-KCNV2 Opti expression constructs, respectively. B shows FACS data showing the percentage of Kv8.2-Alexa488 positive cell population in non-transfected control versus cells transfected with pCAG-KCNV2 WT or pCAG-KCNV2 Opti expression constructs, respectively. There was no significant difference in % Kv8.2 positive cells between the codon-optimized (Opti) and wild-type (WT) vectors. Figure 1 shows the transduction efficiency in transduced ARPE19 cells. A shows Kv8.2 fluorescence intensity (mean integrated density per cell) in Kv8.2 immunolabeled ARPE19 cells transfected with the indicated AAV5 vectors at two multiplicities of infection (MOIs). B shows the percentage of DAPI-positive ARPE19 cells that were Kv8.2 positive after transduction with the indicated AAV5 vectors at two MOIs. Morphology of retinal organoids. Live brightfield imaging of whole retinal organoids. Typical morphology of transduced WT (top) and KCNV2 KO retinal organoids at day 140. Retinal organoids are layered and contain photoreceptor outer segments ("brush border") and occasional clusters of retinal pigment epithelium (RPE). No gross morphological differences were observed between WT and knockout (KO) retinal organoids. Figure 1 shows transgenic Kv8.2 expression in KCNV2 KO retinal organoids (K28D5). Confocal tile scan analysis 3 weeks after transduction with AAV7m8. Signal is detected in the outermost photoreceptor cell layer. Scale bar = 100 μm and 10 μm. AAV7m8 transduction of inner retinal cells. Transduced retinal cryosections co-stained for Kv8.2 and rod bipolar cell marker PKCa. WT organoids contained some Kv8.2-positive inner retinal cells (arrows) in the inner nuclear layer (INL) (separated from the ONL by a dashed line) in addition to the outer nuclear layer (ONL), but the majority of Kv8.2-positive transduced cells were in the outer nuclear layer. The pRK-KCNV2 vector produced little detectable Kv8.2 protein (lower panel), although a few Kv8.2-positive photoreceptor cells could be seen in the ONL in the AAV7m8 RK-KCVN2 Opti condition (*). Scale bar = 10 μm. AAV transduction of RPE cells is shown. In addition to photoreceptors, RPE cells are present in the organoids. RPE are present as clusters (arrows) rather than flat sheets adjacent to photoreceptor outer segments as seen in vivo. RPE are polarized, expressing CRALBP at their apical surface (A) and nuclei located basally (B). High levels of Kv8.2 are detected throughout the cytoplasm in RPE cells of retinal organoids efficiently transduced with AAV. In contrast, AAV RK-KCNV2 did not result in detectable levels of Kv8.2 expression in RPE cells. Transduction of Müller glial cells with AAV5 pCAG-KCNV2-Opti, AAV5 pRK-KCNV2-Opti, AAV7m9 pCAG-KCNV2-Opti, and AAV7m9 pRK-KCNV2-Opti are shown. CRALBP stains Müller glial cells that span the neural retina and form the outer limiting membrane through tight junctions with rod and cone cells. Müller glia did not co-stain for Kv8.2 despite their close proximity to photoreceptor cells. Figure 1 shows that transgenic Kv8.2 expression is localized to the plasma membrane and photoreceptor inner segments. Clone K28 (Differentiation 5) transduced with 7m8 AAV vector with both codon-optimized KCNV2 vector driven by the CAG promoter and WT KCNV2 vector (CAG KCNV2-WT and 7m8 CAG KCVN2 Opti) stained for rhodopsin and Kv8.2. Nuclei are counterstained with DAPI. Colocalization of Kv8.2 and Kv.2.1 in photoreceptor inner segments. High magnification confocal microscopy of WT organoids versus transduced KCNV2 KO organoids transduced with AAV5 CAG-WT or AAV5 CAG-Opti vectors. The potassium channel Kv2.1 localizes to the plasma membrane of the spherical inner segment structures, and vector-derived Kv8.2 is co-expressed within the inner segment with an expression pattern similar to WT. TUNEL staining in WT, control, and transduced retinal organoids. Whole-body confocal tile scans (40x magnification) of WT retinal organoid cryosections stained with DAPI and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), an indicator of apoptosis. TUNEL reactivity was scant to absent in the retinal cell layers (INL and ONL), but could be detected in the center of the organoid (dashed area) and in areas of non-retinal tissue. TUNEL staining in WT, control, and transduced retinal organoids is shown. Qualitatively, there was no increase in TUNEL reactivity in KCNV2 KO organoids compared to WT, and no increase in AAV7m8-transduced KO organoids. TUNEL staining in WT, control, and transduced retinal organoids is shown. Qualitatively, there was no increase in TUNEL reactivity in KCNV2 KO organoids compared to WT, and no increase in KO organoids transduced with AAV5 vectors containing WT or codon-optimized KCNV2. Cone cell counts in AAV-transduced retinal organoids. LM opsin staining was used to determine the average number of cones per 100 μm in WT, KCNV2 KO, and organoids transduced with the indicated vectors. Each point represents one transduced organoid where cones were counted from one 7 μm retinal cryosection. Whole organoids were imaged at 40x magnification and the number of cones per μm was counted (between 34 and 483 were counted per organoid). All organoids had a good distribution of cone cells. Non-transduced KCNV2 KO organoids had significantly more cones than WT unedited controls (p=0.004, unpaired t-test). AAV transduction (all vectors grouped together) did not reduce cone number compared to WT (p=0.2), but did reduce cone number compared to non-transduced (p=0.02). Error bars = standard deviation Figure 1 shows relative mRNA levels in transduced retinal organoids as determined by qPCR. A shows KCNV2 expression in KCNV2 KO clones K5, K12, and K28 transduced with different AAV vectors containing either a photoreceptor-specific RK or constitutive CAG promoter driving expression of two different versions of the KCNV2 gene: codon-optimized or WT. Untreated KO KCNV2 clones and a WT isogenic control, I5, are included for comparison. The graph shows the expression of codon-optimized KCNV2 21 days after transduction with different AAV vectors, i.e., AAV5-CAG-KCNV2opti, AAV7m8-CAG-KCNV2opti, AAV5-RK-KCNV2opti, or AAV7m8-RK-KCNV2-Opti. Results are expressed as fold change in KCNV2 mRNA expression relative to the lowest expressing sample (AAV5 CAG-KCNV2-Opti). B is a graph showing expression of WT KCNV2 21 days after transduction with the indicated AAV vectors. Results are expressed as fold change in WT KCNV2 mRNA relative to age-matched non-transduced controls derived from the same KO clones. Quantification of retinal organoid immunofluorescence is shown. Total Kv8.2 fluorescence in the outer nuclear layer (ONL) is shown. Bars = mean fluorescence of transduced organoids normalized to the area measured, and expressed as % fluorescence in WT control organoids. Tile scan images were acquired to obtain fluorescence measurements over the entire length of the ONL. Dotted lines represent the mean "background" signal in non-transduced KO control organoids. Each point represents one organoid. n = 3-4 organoids from independent experiments. Error bars = +/- SEM. There was a significant difference in total fluorescence between the CAG and RK promoters in both 7m8 and AAV5 capsids (p = 0.031 and 0.028, respectively, two-tailed, paired Student's t-test). Despite a trend towards increased fluorescence intensity in codon-optimized (Opti) vectors, there was no significant difference between WT and Opti in vectors containing CAG or RK promoters. Quantification of retinal organoid immunofluorescence. Representative immunofluorescence in the photoreceptor layer of transduced KCNV2 KO, WT, and AAV7m8 Kv8.2 transduced organoids. Kv8.2 and potassium channel subunit Kv.2.1, cone arrestin (Arr3), and nuclei are stained with DAPI. Scale bar = 10 μm. Relative colocalization of Kv8.2 and Kv2.1 in transduced organoids is shown. Organoid cryosections were co-stained with Kv.2.1 and Kv8.2, and the total colocalized area was measured on thresholded images in FIJI and normalized to the length of the retina assayed per organoid. Results are expressed as fold change compared to non-transduced controls. Results were analyzed by one-way ANOVA and Dunnett's multiple comparison test. *p=0.02, **p=0.002. Proximity ligation assay (PLA) signal specificity in transduced organoids. PLA signals (dots) after Kv2.1 and Kv8.2 co-staining were abundant in the peripheral retinal organoids where the photoreceptor inner/outer segments are located. Figure 1 shows proximity ligation assay (PLA) signal specificity in transduced organoids. PLA signal was almost absent in the ONL (photoreceptor layer) of KCNV2 KO organoids. Proximity ligation assay (PLA) signal specificity in transduced organoids. Quantification of PLA puncta (imaged J) normalized to the measured area per organoid (n=3 regions of interest (ROIs)). PLA signal was significantly reduced (p<0.03) in KCNV2 KO photoreceptors compared to WT (clones K28 and K12). Error bars=SEM. Figure 1 shows PLA signal in AAV5 and AAV7m8 transduced photoreceptors. 63x maximum intensity projection from 7 μm organoid cryosection. The photoreceptor layer (ONL) has a distinctive dense structure seen in the DAPI channel above the outer plexiform layer without nuclei. PLA signal (dots) indicates Kv.2.1/Kv8.2 protein-protein interaction. PLA signal was concentrated at the apical end of the ONL in the region of the photoreceptor inner segment (IS). Transduced organoids had higher PLA signal density than non-transduced KCNV2 KO organoids derived from IPSC clone K12. Quantification of PLA puncta in transduced retinal organoid clones ((A) Clone 12, (B) Clone 28). Bars represent the average number of PLA puncta in the ONL per field normalized to the area measured (approximately 32 per 100-150 photoreceptors, 10-600 puncta counted per field). Error bars = SEM).

Provided herein are expression constructs, viral genomes, and vectors for expression of potassium voltage-dependent channel modulator subfamily V member 2 (Kv8.2), as well as methods of using the expression constructs, viral genomes, and vectors to treat retinal diseases associated with one or more mutations in the KCNV2 gene.

Kv8.2
Kv8.2 is a voltage-gated potassium channel subunit. The KCNV2 gene is located on chromosome 9p24.2 and contains two exons that code for the 545 amino acid Kv8.2 protein. Kv8.2 cannot form functional homomeric channels, but interacts with other potassium channel subunits Kv2.1 and Kv2.2, altering their biophysical properties. Kv8.2 is the only silent subunit that has been implicated in human disease so far. Variants/mutations cause a severe hereditary photoreceptor dystrophy known as "cone dystrophy with supernormal rod response" (CDSSR).

KCNV2 (Kv8.2) is expressed in the inner segments (ellipsoid and myoid regions) of rod and cone photoreceptors in the retina but is absent in the outer segments of humans, mice, and macaques. Kv8.2 interacts with Kv2.1, which is expressed in rod and cone inner segments, and Kv2.2, which is expressed in human cones but not rods.

KCNV2 (Kv8.2) homozygous knockout (KO) mice show many similarities to the human disorder, including electroretinograms (ERGs) with reduced a-wave and elevated b-wave responses to bright light stimuli. KCNV2 KO mice show reduced cone cell numbers (80% of WT), increased TUNEL-positive cells throughout the retina (1, 3, and 6 months of age), and general thinning of the outer nuclear layer (ONL, 60% of WT at 6 months of age).

The precise subcellular localization of many important photoreceptor proteins has been previously demonstrated (e.g., Rhodopsin, RetGC, ABCA4 are located in rod outer segments, Bassoon, Ribeye are located in synaptic terminals). Although the presence of KCNV2 transcripts has been detected in human retinal organoids by single-cell RNA-seq, the presence of potassium channel subunits Kv8.2, Kv2.1, and Kv2.2 in human retinal organoids derived from human embryonic stem cells (HESCs) or induced pluripotent stem cells (IPSCs) has not yet been investigated in any publication. Species-specific differences in the function of Kv8.2 and its binding partners (e.g., absence of Kv2.2 in mouse retina) have been reported, making the use of human cell models important for the development of potential KCNV2 AAV gene therapy.

Mutations in KCNV2 can cause retinal diseases, including photoreceptor dystrophies, such as cone dystrophy with supernormal rod response (CDSSR). The diagnosis of such diseases is established by electrophysiological evaluation; the functional outcome depends on the stage of the disease and the age of the individual. For example, CDSSR is associated with an electroretinogram (ERG) with a reduced a-wave and elevated b-wave response to bright light stimuli. The diagnosis of cone dystrophy can be supported by demonstration of a reduced number of cone cells (approximately 80% of normal). For example, retinas with abnormal KCNV2 expression may have an increased number of TUNEL-positive cells and a generalized thinning of the outer nuclear layer (ONL, 60% of normal).

Expression Constructs In one aspect, an expression construct is provided that includes (a) a promoter sequence that confers expression in photoreceptor cells, and (b) a nucleic acid sequence encoding potassium voltage-dependent channel modulator subfamily V member 2 (Kv8.2); the nucleic acid sequence is operably linked to the promoter. As used herein, "operably linked" refers to both expression control sequences (e.g., promoters) that are contiguous with the coding sequence of Kv8.2, and expression control sequences that act in trans or at a distance to control the expression of Kv8.2. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and, if desired, sequences that enhance protein processing and/or secretion.

Numerous expression control sequences, e.g., natural, constitutive, inducible and/or tissue-specific sequences, are known in the art and can be utilized to drive expression of genes, depending on the type of expression desired. For eukaryotic cells, expression control sequences typically include promoter sequences, enhancer sequences, and polyadenylation sequences that may include splice donor and splice acceptor sites. A polyadenylation (polyA) sequence is generally inserted after the sequence encoding Kv8.2 and before the 3'ITR sequence. Another regulatory component of rAAV useful in the methods disclosed herein is an internal ribosome entry site (IRES). IRES sequences can be used to produce multiple polypeptides from a single gene transcript. IRES (or other suitable sequences) can be used to produce proteins containing multiple polypeptide chains or to express two different proteins from or within the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports expression of transgenes in photoreceptors, RPE and ganglion cells. Preferably, the IRES is located 3' to the sequence encoding Kv8.2 in the rAAV vector.

In one embodiment, the promoter sequence comprises a rhodopsin kinase (RK) promoter sequence. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In one embodiment, the promoter sequence comprises SEQ ID NO:7.

In one embodiment, the promoter sequence comprises a synthetic cytomegalovirus-derived promoter sequence (CAG). In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8. In one embodiment, the promoter sequence comprises SEQ ID NO:8.

In some embodiments, the promoter is specific to photoreceptor cells, i.e., the promoter is active in photoreceptor cells but has reduced or no activity in other cell types.

In one embodiment, the nucleic acid sequence encoding Kv8.2 is a coding sequence from the WT KCNV2 gene. In some embodiments, the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. In one embodiment, the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO:9.

In one embodiment, the nucleic acid sequence encoding Kv8.2 is a codon-optimized gene sequence. In some embodiments, the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In one embodiment, the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO: 10.

In some embodiments, the nucleic acid sequence encoding Kv8.2 encodes a protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding Kv8.2 encodes a protein comprising SEQ ID NO: 13.

In one embodiment, the expression construct comprises a post-transcriptional regulatory element. In one embodiment, the expression construct comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11. In one embodiment, the post-transcriptional regulatory element comprises SEQ ID NO:11.

In one embodiment, the expression construct comprises a polyadenylation signal. In one embodiment, the expression construct comprises a bovine growth hormone polyadenylation (BGH-polyA) signal. In some embodiments, the polyadenylation signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12. In one embodiment, the polyadenylation signal comprises SEQ ID NO:12.

Vector In one aspect, a recombinant vector and its use for introducing a transgene or expression construct into a cell are provided. In some embodiments, the recombinant vector comprises a recombinant DNA construct that includes additional DNA elements, including a DNA segment that results in the replication of DNA in a host cell and the expression of a target gene in the target cell at an appropriate level. Those skilled in the art will understand that expression control sequences (promoters, enhancers, etc.) are selected based on their ability to promote the expression of a target gene in a target cell. As used herein, "vector" refers to a vehicle that includes a polynucleotide to be delivered to a host cell in vitro or in vivo. Non-limiting examples of vectors include recombinant plasmids, yeast artificial chromosomes (YACs), minichromosomes, DNA minicircles, or viruses (including sequences derived from viruses). A vector may also refer to a virion that includes a nucleic acid that is delivered to a host cell either in vitro or in vivo. In some embodiments, a vector refers to a virion that includes a recombinant viral genome, where the recombinant viral genome includes one or more ITRs and a transgene.

In one embodiment, the recombinant vector is a viral vector or a combination of multiple viral vectors.

In one aspect, a vector is provided that includes any of the expression constructs disclosed herein.

In one aspect, a vector is provided that includes a nucleic acid comprising (a) a promoter sequence that confers expression in a photoreceptor cell, and (b) a nucleic acid sequence that encodes Kv8.2, the nucleic acid sequence encoding Kv8.2 being operably linked to the promoter.

In one embodiment, the promoter sequence comprises a rhodopsin kinase (RK) promoter sequence. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In one embodiment, the promoter sequence comprises SEQ ID NO:7.

In one embodiment, the promoter sequence comprises a synthetic cytomegalovirus-derived promoter sequence (CAG). In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8. In one embodiment, the promoter sequence comprises SEQ ID NO:8.

In some embodiments, the promoter is specific to photoreceptor cells.

In one embodiment, the nucleic acid sequence encoding Kv8.2 is a coding sequence from the WT KCNV2 gene. In some embodiments, the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. In one embodiment, the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO:9.

In one embodiment, the nucleic acid sequence encoding Kv8.2 is a codon-optimized gene sequence. In some embodiments, the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In one embodiment, the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO: 10.

In some embodiments, the nucleic acid sequence encoding Kv8.2 encodes a protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding Kv8.2 encodes a protein comprising SEQ ID NO: 13.

In one embodiment, the vector comprises a nucleic acid comprising a post-transcriptional regulatory element. In one embodiment, the vector comprises a nucleic acid comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11. In one embodiment, the post-transcriptional regulatory element comprises SEQ ID NO:11.

In one embodiment, the vector comprises a nucleic acid comprising a polyadenylation signal. In one embodiment, the vector comprises a nucleic acid comprising a bovine growth hormone polyadenylation (BGH-polyA) signal. In some embodiments, the polyadenylation signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12. In one embodiment, the polyadenylation signal comprises SEQ ID NO:12.

In one embodiment, the vector comprises a nucleic acid comprising one or more inverted terminal repeats (ITRs). In one aspect, the ITR sequence is derived from AAV serotype 2. In one embodiment, the 5'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5. In one embodiment, the 5'ITR sequence comprises SEQ ID NO:5. In one embodiment, the 3'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6. In one embodiment, the 3'ITR sequence comprises SEQ ID NO:6.

In some embodiments, the vector comprises a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 1-4.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising the RK promoter sequence;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter;
(c) WPRE;
(d) a BGH-polyA signal; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a CAG promoter sequence;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter;
(c) WPRE;
(d) a BGH-polyA signal; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising the RK promoter sequence;
(b) a codon-optimized nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter;
(c) WPRE;
(d) a BGH-polyA signal; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a CAG promoter sequence;
(b) a codon-optimized nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter;
(c) WPRE;
(d) a BGH-polyA signal; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7;
(b) a nucleic acid sequence encoding a Kv8.2 protein, wherein the Kv8.2 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, and wherein the nucleic acid sequence encoding the Kv8.2 protein is operably linked to a promoter;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8;
(b) a nucleic acid sequence encoding a Kv8.2 protein, wherein the Kv8.2 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, and wherein the nucleic acid sequence encoding the Kv8.2 protein is operably linked to a promoter;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:7;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO:9;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:8;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO:9;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:7;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO: 10;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:8;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO: 10;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:7;
(b) a nucleic acid sequence encoding a Kv8.2 protein, wherein the Kv8.2 protein comprises SEQ ID NO: 13, and wherein the nucleic acid sequence encoding the Kv8.2 protein is operably linked to a promoter;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:8;
(b) a nucleic acid sequence encoding a Kv8.2 protein, wherein the Kv8.2 protein comprises SEQ ID NO: 13, and wherein the nucleic acid sequence encoding the Kv8.2 protein is operably linked to a promoter;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

Viral Vectors Viral vectors for expressing a target gene in a target cell, tissue, or organism are known in the art and include, for example, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, poxvirus vectors, baculovirus vectors, herpes simplex virus vectors, vaccinia virus vectors, or synthetic viral vectors (e.g., chimeric, mosaic, or pseudotyped viruses, and/or viruses containing foreign proteins, synthetic polymers, nanoparticles, or small molecules).

AAV Vector Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires a helper virus to promote efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames that code for Rep and Cap proteins, respectively. The Rep reading frame codes for four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. These proteins mainly function in AAV replication and rescue and in controlling AAV integration into host cell chromosomes. The Cap reading frame codes for three structural proteins with molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the virion capsid. More than 80% of the total protein in the AAV virion is composed of VP3. Adjacent to the 5' and 3' ends of the rep and cap open reading frames are ITRs approximately 141 bp in length. The ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with therapeutic or reporter transgenes.

Recombinant adeno-associated virus "rAAV" vectors include any vector derived from any adeno-associated virus serotype. rAAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the Rep and/or Cap genes, but retain functional flanking ITR sequences.

In some embodiments, the viral vector is a rAAV virion that comprises a rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome comprises an expression cassette disclosed herein.

In some embodiments, the viral vectors disclosed herein comprise a nucleic acid that includes the AAV 5'ITR and 3'ITR located 5' and 3', respectively, to the sequence encoding Kv8.2. However, in certain embodiments, it may be desirable for the nucleic acid to contain 5'ITR and 3'ITR sequences arranged in tandem, e.g., 5'-3', or head-to-tail, or in another alternative arrangement. In still other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITRs, or to have the 5'ITR (or conversely, the 3'ITR) located both 5' and 3' of the sequence encoding Kv8.2. The ITR sequences may be located immediately upstream and/or downstream of the heterologous molecule, or intervening sequences may be present. The ITRs need not be wild-type nucleotide sequences, and may be modified (e.g., by insertion, deletion, or substitution of nucleotides), so long as the sequences provide functional rescue, replication, and packaging. The ITRs can be selected from AAV2 or from other AAV serotypes, as described herein.

In some embodiments, the viral vector is an AAV vector, such as, for example, AAV1 (i.e., an AAV containing AAV1 ITRs and AAV1 capsid proteins), AAV2 (i.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., an AAV8 AAVrh. ITR and AAV8 capsid protein), AAV9 (i.e., AAV containing AAV9 ITR and AAV9 capsid protein), AAVrh74 (i.e., AAV containing AAVrh74 ITR and AAVrh74 capsid protein), AAVrh. 8 (i.e., AAV containing AAVrh.8 ITR and AAVrh.8 capsid protein), or AAVrh. 10 (i.e., AAV containing AAVrh.10 ITR and AAVrh.10 capsid protein).

In some embodiments, the viral vector is a pseudotyped AAV vector that includes ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2/5 (i.e., an AAV that contains AAV2 ITRs and AAV5 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2/7m8 (i.e., an AAV that contains AAV2 ITRs and AAV7m8 capsid proteins).

In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein that contains a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In embodiments, the capsid is a variant AAV capsid, such as the AAV2 variant rAAV2-retro (SEQ ID NO:44 from WO2017/218842, incorporated herein by reference).

In one aspect, a viral genome is provided that includes a nucleic acid comprising: (a) a promoter sequence that confers expression in a photoreceptor cell; and (b) a nucleic acid sequence that encodes Kv8.2, the nucleic acid sequence encoding Kv8.2 being operably linked to the promoter.

In one embodiment, the promoter sequence comprises an RK promoter sequence. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In one embodiment, the promoter sequence comprises SEQ ID NO:7.

In one embodiment, the promoter sequence comprises a CAG promoter sequence. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8. In one embodiment, the promoter sequence comprises SEQ ID NO:8.

In some embodiments, the promoter is specific to photoreceptor cells.

In one embodiment, the nucleic acid sequence encoding Kv8.2 is a coding sequence derived from the wild-type KCNV2 gene. In some embodiments, the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. In one embodiment, the nucleic acid sequence encoding RetGC comprises SEQ ID NO:9.

In one embodiment, the nucleic acid sequence encoding Kv8.2 is a codon-optimized gene sequence. In some embodiments, the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In one embodiment, the nucleic acid sequence encoding RetGC comprises SEQ ID NO: 10.

In some embodiments, the nucleic acid sequence encoding Kv8.2 encodes a protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding Kv8.2 encodes a protein comprising SEQ ID NO: 13.

In one embodiment, the viral genome comprises a nucleic acid comprising a post-transcriptional regulatory element. In one embodiment, the viral genome comprises a nucleic acid comprising WPRE. In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11. In one embodiment, the post-transcriptional regulatory element comprises SEQ ID NO:11.

In one embodiment, the viral genome comprises a nucleic acid comprising a polyadenylation signal. In one embodiment, the viral genome comprises a nucleic acid comprising a BGH-polyA signal. In some embodiments, the polyadenylation signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12. In one embodiment, the polyadenylation signal comprises SEQ ID NO:12.

In one aspect, the viral genome comprises a nucleic acid comprising one or more inverted terminal repeats (ITRs). In one embodiment, the ITR sequence is derived from AAV serotype 2. In one embodiment, the 5'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5. In one embodiment, the 5'ITR sequence comprises SEQ ID NO:5. In one embodiment, the 3'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6. In one embodiment, the 3'ITR sequence comprises SEQ ID NO:6.

In some embodiments, the viral genome comprises a nucleic acid comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the sequences of SEQ ID NOs: 1-4. In some embodiments, the viral genome comprises a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 1-4.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising the RK promoter sequence;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter;
(c) WPRE;
(d) a BGH-polyA signal; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a CAG promoter sequence;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter;
(c) WPRE;
(d) a BGH-polyA signal; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising the RK promoter sequence;
(b) a nucleic acid sequence encoding a codon-optimized Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter;
(c) WPRE;
(d) a BGH-polyA signal; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a CAG promoter sequence;
(b) a nucleic acid sequence encoding a codon-optimized Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter;
(c) WPRE;
(d) a BGH-polyA signal; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7;
(b) a nucleic acid sequence encoding a codon-optimized Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8;
(b) a nucleic acid sequence encoding a codon-optimized Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7;
(b) a nucleic acid sequence encoding a Kv8.2 protein, wherein the Kv8.2 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, and wherein the nucleic acid sequence encoding the Kv8.2 protein is operably linked to a promoter;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8;
(b) a nucleic acid sequence encoding a Kv8.2 protein, wherein the Kv8.2 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, and wherein the nucleic acid sequence encoding the Kv8.2 protein is operably linked to a promoter;
(c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11;
(d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:7;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO:9;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:8;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO:9;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:7;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO: 10;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:8;
(b) a nucleic acid sequence encoding Kv8.2, wherein the nucleic acid sequence encoding Kv8.2 is operably linked to a promoter, and the nucleic acid sequence encoding Kv8.2 comprises SEQ ID NO: 10;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:7;
(b) a nucleic acid sequence encoding a Kv8.2 protein, wherein the Kv8.2 protein comprises SEQ ID NO: 13, and wherein the nucleic acid sequence encoding the Kv8.2 protein is operably linked to a promoter;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following:
(a) a promoter sequence comprising SEQ ID NO:8;
(b) a nucleic acid sequence encoding a Kv8.2 protein, wherein the Kv8.2 protein comprises SEQ ID NO: 13, and wherein the nucleic acid sequence encoding the Kv8.2 protein is operably linked to a promoter;
(c) a post-transcriptional regulatory element comprising SEQ ID NO:11;
(d) a polyadenylation signal comprising the sequence of SEQ ID NO: 12; and (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

Adenovirus (AV) vectors include, for example, those based on human adenovirus type 2 and human adenovirus type 5, which have been rendered replication-deficient by deletion of the E1 and E3 regions. A transcription cassette can be inserted into the E1 region to obtain an E1/E3 deleted recombinant AV vector. Adenovirus vectors include helper-dependent, large-capacity adenovirus vectors (also known as large-capacity, "gutless" or "gutted" vectors) that do not contain viral coding sequences. These vectors contain cis-acting elements required for viral DNA replication and packaging, mainly inverted terminal repeats (ITRs) and packaging signals (CY). These helper-dependent AV vector genomes can carry from a few hundred base pairs up to approximately 36 kb of foreign DNA.

Alternatively, other systems such as lentiviral vectors can be used. Lentiviral-based systems are capable of transducing non-dividing as well as dividing cells, making them useful for applications targeting non-dividing cells of the CNS, for example. Lentiviral vectors are derived from the human immunodeficiency virus and, like that virus, integrate into the host genome, offering the potential for very long-term gene expression.

Polynucleotides, including plasmids, YACs, minichromosomes and minicircles carrying target gene containing expression cassettes, can also be introduced into cells or organisms by non-viral vector systems using, for example, cationic lipids, polymers, or both as carriers. Conjugated poly-L-lysine (PLL) polymer and polyethyleneimine (PEI) polymer systems can also be used to deliver vectors to cells. Other methods of delivering vectors to cells include the use of hydrodynamic injection, electroporation, and ultrasound for both cell cultures and organisms. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, N. et al. (Adv Biomed Res. 2012;1:27), which is incorporated herein by reference.

rAAV virion production The rAAV virions disclosed herein can be constructed and produced using the materials and methods described herein and those known to those skilled in the art. Such engineering methods used to construct any embodiment of the present invention are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al. and Ausubel et al. cited above; and International Patent Publication No. WO95/13598. Additionally, suitable methods for producing rAAV cassettes in adenovirus capsids are described in U.S. Patent Nos. 5,856,152 and 5,871,982.

Briefly, in order to package the rAAV genome into rAAV virions, the host cell must contain sequences necessary to express AAV rep and AAV cap or functional fragments thereof, as well as helper genes necessary for AAV production. The AAV rep and cap sequences are obtained from AAV sources found herein. The AAV rep and cap sequences can be introduced into the host cell in any manner known to those skilled in the art, including, but not limited to, transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences can be transfected into the host cell by one or more nucleic acid molecules and stably present in the cell as an episome. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. In another embodiment, the rep and cap sequences are transiently expressed in the host cell. For example, a nucleic acid molecule useful for such transfection includes, from 5' to 3', a promoter, an optional spacer inserted between the promoter and the start of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.

The rep and cap sequences, together with their expression control sequences, may be provided on a single vector, or each sequence may be provided on its own vector. Preferably, the rep and cap sequences are provided on the same vector. Alternatively, the rep and cap sequences may be provided on a vector containing other DNA sequences that may be introduced into the host cell. Preferably, the promoter used in this construct may be any suitable constitutive, inducible, or native promoter known to those skilled in the art. The molecule providing the rep and cap proteins may be in any form that delivers these components to the host cell. Desirably, this molecule is in the form of a plasmid and may contain other non-viral sequences, such as sequences of marker genes. This molecule does not contain AAV ITRs and generally does not contain AAV packaging sequences. Other viral sequences, particularly adenoviral sequences, are avoided in this plasmid to avoid the occurrence of homologous recombination. This plasmid is desirably constructed so that it can be stably transfected into cells.

Molecules providing rep and cap can be transiently transfected into host cells, but it is preferred that the host cells are stably transformed with the sequences necessary to express functional rep/cap proteins in the host cells, for example as episomes or by integration into the host cell chromosome. Depending on the promoter controlling expression in such stably transfected host cells, the rep/cap proteins can be expressed transiently (e.g., through the use of an inducible promoter).

The methods used to construct embodiments of the invention are conventional genetic or recombinant engineering techniques, such as those described in the references above. For example, rAAV can be generated utilizing a triple transfection method using either the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. See Herzog et al, 1999, Nature Medic., 5(1):56-63 for the method used in the following examples, which uses a plasmid carrying the transgene, a helper plasmid containing CPA-RPE65, AAV rep and cap, and a plasmid supplying the adenoviral helper functions of E2A, E4Orf6, and VA. Although the present specification provides examples of specific constructs using the information provided herein, one of skill in the art can select and design other suitable constructs using the selection of spacers, promoters, and other elements including at least one translation start and stop signal, and the optional addition of polyadenylation sites.

rAAV virions are then produced by culturing host cells containing the rAAV virus described herein, containing the rAAV genome, AAV rep sequences and AAV cap sequences to be packaged into rAAV virions, under the control of regulatory sequences that direct their expression. Suitable viral helper genes, such as adenovirus E2A, E4Orf6 and VA, among other possible helper genes, can be provided to the culture by various methods known in the art, preferably on separate plasmids. Recombinant AAV virions that direct expression of the transgene are then isolated from the cells or cell culture in the absence of contaminating helper virus or WT AAV.

Expression of the KCNV2 gene can be measured by methods known in the art. For example, target cells can be infected in vitro and the number of transgene copies in the cells can be monitored by Southern blotting or quantitative polymerase chain reaction (PCR). RNA expression levels can be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR (qPCR); and protein expression levels can be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or by specific methods detailed in the Examples below.

Pharmaceutical Compositions Provided herein are pharmaceutical compositions comprising any of the vectors disclosed herein and a pharma- ceutically acceptable excipient.

The recombinant AAV containing the gene encoding Kv8.2 is preferably evaluated for contamination by conventional methods and then formulated into a pharmaceutical composition suitable for administration to a patient.

Such formulations involve the use of a pharma- ceutically and/or physiologically acceptable vehicle or carrier, particularly a vehicle or carrier suitable for subretinal injection, such as buffered saline or other buffers, e.g., HEPES, to maintain the pH at an appropriate physiological level.

The vectors of the invention can be formulated into pharmaceutical compositions. These compositions may contain, in addition to the vector, pharma- ceutically and/or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials can be determined by the skilled artisan according to the route of administration. Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Additional carriers are provided in International Patent Publication No. WO 00/15822, which is incorporated herein by reference. Physiological saline, magnesium chloride, dextrose, or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol, may be included. In some cases, surfactants may be used, such as pluronic acid (PF68) 0.001%. In some cases, Ringer's solution, lactated Ringer's solution, or Hartmann's solution is used. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as required.

For delayed release, the vector may be included in a pharmaceutical composition formulated for sustained release, such as in a microcapsule formed from a biocompatible polymer or a liposomal carrier system by methods known in the art.

For long-term storage, the virus may be frozen in the presence of glycerol.

Method of Treatment Provided herein is a method of treating retinal disease in a subject in need of treatment, wherein the retinal disease is associated with one or more mutations in KCNV2 gene, comprising administering to the subject a vector disclosed herein. Provided herein is a vector for use in a method of treating retinal disease in a subject in need of treatment, wherein the retinal disease is associated with one or more mutations in KCNV2 gene. In some embodiments, the subject has a mutation in KCNV2.

In some embodiments, the subject is a mammal. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, household pets, and farm animals. Mammals include, but are not limited to, humans, or non-human mammals, such as, for example, cows, horses, dogs, sheep, or cats. Individuals and patients are also of interest herein.

As used herein, the terms "treat," "treated," "treating," or "treatment" refer to therapeutic treatment, the purpose of which is to slow (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. For purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder, or disease; stabilization (i.e., not worsening) of the pathological condition, disorder, or disease state; delay in onset of the condition, disorder, or disease, or delay in progression of the condition, disorder, or disease; amelioration of the condition, disorder, or disease state; and remission (partial or complete), or improvement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment. The terms "prevent", "prevention" and the like refer to acting before the onset of an obvious disease or disorder, preventing a disease or disorder from developing, or minimizing the extent of or slowing the progression of a disease or disorder.

In some embodiments, the retinal disease is a cone dystrophy. In one embodiment, the retinal disease is a cone dystrophy with supernormal rod response (CDSSR).

In one aspect, a method is provided that includes:
(a) determining whether the subject carries a mutation in the KCNV2 gene; and (b) if the subject carries a mutation in the KCNV2 gene, administering to the subject a pharmaceutical composition comprising a vector disclosed herein.

Routes and Methods of Administration In some embodiments, the vectors or pharmaceutical compositions disclosed herein are administered by intraocular injection. In some embodiments, the vectors or pharmaceutical compositions disclosed herein are administered by direct retinal injection, subretinal injection, or intravitreal injection. In some embodiments, the vectors or pharmaceutical compositions disclosed herein are administered to the central retina of a subject.

The dose of the vector of the present invention can be determined according to various parameters, in particular the age, weight and condition of the patient to be treated, the specific eye disorder and the extent to which the disorder has progressed if progressive, the route of administration, and the required regimen. A physician can also determine the route of administration and dosage required for a particular patient. An effective amount of rAAV carrying a nucleic acid sequence encoding a desired transgene under the control of a promoter sequence desirably ranges between about 1×10 ⁹ and 2×10 ¹² rAAV genome particles, or between 1×10 ¹⁰ and 2×10 ¹¹ genome particles. A genome particle is defined herein as an AAV capsid containing a single-stranded DNA molecule that can be quantified using a sequence-specific method, such as real-time PCR. In some embodiments, about 1×10 ⁹ to 2×10 ¹² rAAV genome particles are provided in a volume of between about 150 and about 800 μl. In some embodiments, about 1×10 ¹⁰ to 2×10 ¹¹ rAAV genome particles are provided in a volume of between about 250 to about 500 μl. Still other doses within these ranges can be selected by the attending physician.

The dose may be provided as a single dose, but may be repeated for the fellow eye, or if for some reason (such as surgical complications) the vector does not target the correct area of the retina. The treatment is preferably a single permanent treatment for each eye, but repeated injections, for example with a different AAV serotype, may be considered in the future. Thus, it may be desirable to administer multiple "booster" doses of the pharmaceutical composition disclosed herein. For example, depending on the duration of the transgene in the target cells of the eye, booster doses can be delivered at six-month intervals or annually after the initial administration. Such booster doses and the need for them can be monitored by the attending physician, for example, using retinal and visual function tests and visual behavior tests known in the art. Other similar tests may be used to determine the condition of the treated subject over time. Selection of the appropriate test can be performed by the attending physician. Additionally alternatively, the methods disclosed herein may also involve injection of larger amounts of vector-containing solution in single or multiple infections to allow for levels of visual function approaching those seen in WT retina.

Additional Methods In one aspect, there is provided a method of increasing expression of Kv8.2 in a subject in need thereof, comprising administering to the subject a vector as disclosed herein. In one aspect, there is provided a method of increasing expression of Kv8.2 in a cell, comprising contacting the cell with a vector as disclosed herein.

Also provided are kits or articles of manufacture for use in the methods described herein. In an aspect, the kit comprises a composition described herein (e.g., a composition for delivery of a Kv8.2 coding sequence) in suitable packaging. Suitable packaging for the compositions described herein (e.g., ophthalmic compositions for injection) is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may be further sterilized and/or sealed.

Kits containing the compositions described herein are also provided. These kits may further include instruction(s) on how to use the compositions, such as the uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts containing instructions for performing administration of the compositions or for performing any of the methods described herein. For example, in some embodiments, the kit includes an rAAV containing a KCNV2 transgene for expression of Kv8.2 protein in target cells, a pharma- ceutically acceptable carrier suitable for injection, and one or more of a buffer, diluent, filter, needle, syringe, and package insert containing instructions for performing an injection.

It is to be understood that the invention is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention. Moreover, it is to be understood that the disclosure of the invention herein includes all possible combinations of such particular features. For example, if a particular feature is disclosed in connection with a particular aspect or embodiment of the invention, or in a particular claim, that feature can also be used in combination with and/or in connection with other particular aspects and embodiments of the invention, and in the invention generally, to the extent possible.

When reference is made herein to a method that includes two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes this possibility), and the method may include one or more other steps that occur before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes these possibilities).

All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, if the definition or use of a term in a reference incorporated herein by reference is inconsistent with or contradicts the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

To facilitate a better understanding of the present invention, the following examples of specific embodiments are given. The following examples should not be construed as limiting or defining the entire scope of the invention.

Example 1: Design and construction of AAV-KCNV2 expression constructs First, KCNV2 cDNA or codon-optimized KCNV2 cDNA was cloned into the AAV single-stranded backbone downstream of the ubiquitous CAG promoter or the photoreceptor-specific RK1 promoter. A Kozak consensus sequence was placed between the promoter and the transgene. A Woodchuck Hepatitis Virus Variant 6 (WPREm6) sequence was placed between the transgene and polyA. The polyA sequence was the bovine growth hormone polyA (BghpA) sequence. See FIG. 1 for a schematic diagram of the four expression constructs. Cloning was performed at VectorBuilder, Inc. (Chicago, IL, USA). Upon receiving the plasmids, complete sequencing (including the ITR regions) was performed by Genewiz (South Plainfield, NJ, USA) and sequences were aligned to the plasmid map using Snapgene (San Diego, CA, USA). The four constructs were packaged into both AAV5 and 7m8 capsids by triple transfection into HEK293T cells and purified by cesium chloride centrifugation at SignaGen laboratories (Frederick, MD, USA).

Example 2: Validation of CAG expression constructs by transfection in cell lines.
To validate the transgenic expression constructs, HEK293 and developing retinal pigment epithelial (ARPE19) cells were transfected using standard nucleofection techniques. First, cells were transfected with expression constructs (pCAG-KCNV2 WT and pCAG-KCNV2 Opti) containing the WT or codon-optimized KCNV2 gene, respectively, under the control of the CAG promoter. An expression plasmid containing a green fluorescent protein (GFP) transgene and the human cytomegalovirus (CMV) promoter (CMV-GFP) was used as a control. Expression was verified by qPCR, immunofluorescence, and FACS.

Validation of CAG expression constructs by qPCR.
KCNV2 WT and KCNV2 Opti mRNA levels were assessed 48 hours after nucleofection of HEK293 or ARPE19 cells with pCAG-KCNV2 WT or pCAG-KCNV2 Opti expression constructs, respectively. mRNA levels were determined by qPCR using TAQMAN primer probe sets designed to detect WT and Opti transcripts. Expression levels were normalized to the housekeeping genes GAPDH and β-actin. Both expression plasmids produced detectable KCNV2 mRNA in both cell lines. Despite being transfected with the same amount of plasmid DNA, ARPE19 had less both WT and Opti transcripts than HEK293 at 48 hours, suggesting lower transfection efficiency (Figure 2). Due to differences in amplification efficiency between primer pairs, Opti and WT transcript levels cannot be directly compared in this analysis.

Validation of CAG expression constructs by immunofluorescence.
ARPE19 cells were nucleofected with pCAG-KCNV2 WT and pCAG-KCNV2 Opti expression constructs, respectively. ARPE19 cells were also transfected with pmaxGFP (GFP driven by the CAG promoter) expression construct from Lonza Biosciences (Morrisville, NC, USA) as a control. Kv8.2 protein was detected using KCNV2 rabbit polyclonal primary antibody (Sigma Aldrich #HPA031131, 1:100) and donkey anti-rabbit Alexa Fluor555 secondary antibody. Certain ARPE19 cells transfected with both KCNV2 expression plasmids produced Kv8.2 protein detectable by immunofluorescence (Figure 3). Transfection levels were low, as indicated by many ARPE19 cells that had no detectable Kv8.2 protein, which was localized to the cell membrane and cytoplasm.

Validation of CAG expression constructs by fluorescence-activated single cell sorting (FACS) HEK293 cells were transfected with 3.5 μg of pCAG-KCNV2 WT or pCAG-KCNV2 Opti expression constructs and harvested 48 hours later. pCMV-GFP expression construct was used as a control. Cells were stained in suspension with Kv8.2 primary antibody (Sigma Aldrich #HPA031131, 1:100) and Alexa Fluor488 anti-rabbit antibody. Cell populations were gated against non-transfected controls (Figure 4). The number of Kv8.2 expressing cells in WT vs. codon-optimized plasmids was not significantly different at 48 hours (n=3 independent experiments).
Median fluorescence intensity (MFI) was used to quantitate Kv8.2 protein expression levels in transfected cells. There was no significant difference between the median fluorescence of Kv8.2/Alexa Fluor488 stained cells in KCNV2 WT vs. KCNV2 Opti expressing constructs at 48 hours (n=3 independent experiments).

Example 3: AAV transduction of expression constructs in ARPE19 cells. ARPE19 cells were transduced with AAV5 KCNV2 vectors (CAG-KCNV2 WT, CAG-KCNV2 Opti, RK-KCNV2 WT, and RK-KCNV2 Opti) at two multiplicities of infection (MOI) (1E4 vector genomes (VG) per cell and 1E5 VG per cell) on chamber slides and fixed 21 days later. Cells stained with Kv8.2 primary and Alexa Fluor 555 secondary antibodies were imaged confocal. Three images (from triplicate wells) were taken per condition and blinded before analysis in FIJI (Image J). The percentage of Kv8.2 expressing cells was scored against DAPI and the mean staining intensity (integrated density) of Kv8.2 expressing cells was quantified with FIJI (Image J).

CAG promoter-expressing AAV (Opti and WT) had higher scores than RK (Opti and WT) in the percentage of Kv8.2 expressing cells and Kv8.2 staining intensity levels. CAG Opti and CAG WT vectors had high variability but did not have significantly different staining intensity levels in Kv8.2 expressing cells at any MOI. CAG Opti had significantly more Kv8.2 positive cells in the 1E4 condition but not in 1E5 (Figure 5).

Example 4: AAV Transduction of Expression Constructs in Organoids Methods for AAV Transduction of Organoids with Expression Constructs Retinal organoids were transferred to 96-well low attachment plates (one organoid per well) and the retinal organoids were transfected with eight AAV KCNV2 constructs (AAV5 CAG-KCNV2 WT, AAV5 CAG-KCNV2 Opti, AAV5 RK-KCNV2 WT, AAV5 RK-KCNV2 Opti, AAV7m8 CAG-KCNV2 WT, AAV7m8 CAG-KCNV2 Opti, AAV7m8 RK-KCNV2 WT, and AAV7m8 RK-KCNV2 Opti) at a dose of 3E11 viral genomes (VG) per organoid in a total of 100 μL of medium. The next day, organoids were transferred to 24-well low-attachment plates, and medium was changed after 3 days. Retinal organoids for transduction were selected based on morphology; those with clear laminated structures and visible outer segment brush borders (FIG. 6) were selected for fixation and analysis by immunofluorescence. Organoids with internal rosette structures (photoreceptors present in the internal structures) were transduced for qPCR analysis, in which mRNA from all organoids was assayed.

Organoids were cultured for an additional 3 weeks and then harvested by snap freezing of whole organoids (qPCR and Western blot) or fixation in 4% paraformaldehyde (PFA) for 30 min at 4°C. Organoids were then washed twice in standard phosphate-buffered saline (PBS) followed by overnight immersion in 30% sucrose in PBS at 4°C. The next day, organoids were embedded in optimal cutting temperature (OCT) compound and stored at -80°C before cryosectioning at 7 μm.

For each transduction of KCNV2 KO organoids, a non-transduced control and a WT (non-CRISPR edited) control from the same clone and differentiation batch were included.

AAV7m8 KCNV2 transduction in the outermost layer of photoreceptor cells Three weeks after AAV transduction, KCNV2 KO retinal organoids were sectioned and assayed for the transgenic KCNV2 protein product Kv8.2. Confocal analysis of whole retinal organoids revealed that both KCNV2 WT and KCNV2 codon-optimized vectors were expressed in the outermost layer of photoreceptors ( FIG. 7 ).

AAV7m8 KCNV2 transduction of inner retinal cells There was little detectable Kv8.2 product in the inner retinal layers in the transduced organoids. Co-staining with the bipolar cell marker PKCa revealed the absence of Kv8.2 stained PKCa positive bipolar cells, whereas WT retinal organoids had some inner retinal cells immunopositive for Kv8.2 (white arrows in Fig. 7), likely amacrine cells, horizontal cells or cone bipolar cells. In contrast, transduced KCNV2 KO retinal organoids had few Kv8.2 positive inner retinal cells (Fig. 8), despite high expression in transduced photoreceptors in the CAG promoter-containing vector, indicating inaccessibility of AAV to these layers and/or preferential vector tropism to photoreceptor cells.

AAV7m8 KCNV2 Transduction of Retinal Pigment Epithelial (RPE) Cells Pigmented RPE cells and photoreceptors originate from the same developmental progenitor cell population. In vivo, the RPE monolayer resides adjacent to the photoreceptor outer segments that define the border of the subretinal space. RPE cells within retinal organoids are arranged in clusters on the outer surface of the organoid (Figure 9, left panel). When present, RPE cells transduced with both AAV5 and 7m8, CAG-KCVN2 expressed high levels of Kv8.2 protein. RK-KCNV2 did not express detectable Kv8.2 in RPE cells, likely due to the photoreceptor specificity of the RK promoter.

AAV7m8 KCNV2 transduction of Müller glial cells Müller glial cells span the entire thickness of the retina, providing structural support and forming the external and internal limiting membranes. In addition to RPE cells, CRALBP is a marker of Müller glia in retinal organoids, which is found to span the internal and external nuclear layers and form the external limiting membrane. Co-staining of CRALBP with Kv8.2 does not show co-staining of these two markers, suggesting that AAV5 and AAV7m8 transduce and/or express transgenes in Müller glial cells (Figure 10).

Example 5: Localization of Kv8.2 in AAV7m8 KCNV2-transduced cells Localization of Kv8.2 in photoreceptor inner segments Endogenous KCNV2 (Kv8.2) protein has been reported to be expressed at the plasma membrane of rod and cone inner segments, but not in outer segments. Trafficking of photoreceptor proteins to the correct intracellular compartment is important for their function, and mis-trafficking of misfolded proteins underlies the pathogenesis of many inherited retinal degenerative disorders.

Transduced retinal organoids were stained with rhodopsin and found to be precisely localized to the membranous outer segment structures. Transgenic kv8.2 (7m8 CAG-WT and 7m8 CAG-Opti) was found to be localized to the inner segment (IS) and plasma membrane of the photoreceptor cell body (Figure 11). Kv8.2 staining was absent from the outer segment (OS). This suggests that proteins produced from both the WT and codon-optimized vectors are properly transported post-translationally.

Colocalization of Kv8.2 and Kv2.1 in AAV7m8 KCNV2-transduced cells The KCVN2 gene product Kv8.2 interacts with the potassium channel subunit Kv2.1 in the retina. Kv8.2 is a silent Kv channel subunit and can therefore only function through its interaction with larger Kv channel subunits. In WT retinal organoids, the aKv2.1 antibody clearly labeled the photoreceptor inner segments with a stronger signal in the cone inner segments (ellipsoid region) (Figure 12). Endogenous Kv8.2 protein (Figure 12) was present in rod and cone inner segments colocalizing with Kv2.1. In KO retinal organoids, where Kv8.2 is absent, an inner segment ellipsoid pattern of Kv2.1 staining is detected in the photoreceptors. AAV-derived Kv8.2 protein (both WT and codon-optimized vectors) was also expressed in the photoreceptor inner segment structures of transduced retinal organoids, indicating that both transgenes were translated into protein and efficiently transported to the correct intracellular compartment ( FIG. 12 ).

Example 6: Assessment of rescue and toxicity following AAV transduction:
Increased TUNEL reactivity throughout the retina has been reported in KCNV2 KO mouse models at 1, 3, and 6 months of age, with a reduction in cone cell numbers per mm2 to 80% of WT at 6 months of age. To determine whether our prenatal KCNV2 KO retinal cell model recapitulates these phenotypes upon transduction and to assess any vector-associated cytotoxicity, TUNEL reactivity and cone cell numbers were measured in WT versus KO organoids and in KO organoids transduced with all AAV vectors.

TUNEL reactivity in AAV-transduced organoids TUNEL is a method for detecting DNA fragmentation by labeling 3'-hydroxyl ends in double-stranded DNA breaks generated during apoptosis. TUNEL reactivity in retinal organoid cryosections was assessed in KCNV2 KO transduced organoids versus non-transduced controls and WT.

Figure 13A shows TUNEL staining of AAV5 CAG-KCNV2-Opti-treated retinal organoids (clone K28) 3 weeks after transduction. TUNEL-positive cells were mainly observed in the center of the organoid (dashed line), with no or very few TUNEL-positive cells in the retinal cell layers (ONL, INL). There was no increase in TUNEL reactivity of KCNV2 KO photoreceptors compared to WT, suggesting that no "in vitro" retinal degeneration has occurred at this time point in this model.

Neither of the two AAV serotypes induced significant levels of TUNEL-positive cells in the ONL or INL with either the WT or codon-optimized transgenes (Figures 13b and 13c), suggesting that the tested AAV serotypes and overexpressed transgenic proteins were not cytotoxic to retinal cells. The presence of TUNEL-positive cells in the center of the organoids has also been widely reported in other HIPSC retinal organoid models and is most likely due to hypoxia in the center of the retinal organoids and/or insufficient nutrient delivery to the cells in the center.

Clonal cell counts in AAV-transduced organoids KCNV2 KO mice show mild loss of cone cells at 6 months of age, down to 80% of WT levels. To determine whether this phenotype is recapitulated in human fetal retinal organoids, cone cells per 100 μm of retinal tissue in WT and KCNV2 KO retinal organoids were quantified by immunofluorescence. The number of L/M opsin-positive cone cells was counted from tile scans (7 μm retinal cryosections) of the entire organoid taken at 40x magnification and normalized to the total length of retinal tissue. The average number of cone cells was counted in a total of 12 WT and 8 untreated KO retinal organoids. There was a significant increase in the number of cone cells in the KCNV2 KO cell line compared to WT (Figure 14). Transduced retinal organoids (all vectors grouped n=30) showed no statistically significant difference between WT and transduced organoids (p=0.2), but showed a significant reduction compared to non-transduced KOs (p=0.02).

Example 7: Quantitative assessment of transgenic KCNV2 mRNA and Kv8.2 protein in transduced organoids.
qPCR assessment of KCNV2 mRNA levels in transduced organoids Quantitative comparison of vector-driven transgene expression was performed by qPCR. KCNV2 mRNA expression levels were assessed in KCNV2 KO organoids transduced with WT and codon-optimized versions of the KCNV2 gene driven by the RK or CAG promoter and delivered by either AAV2/5 or AAV2/7m8. Whole transduced organoids from clones K12, K5 and K28 were harvested 21 days after transduction by snap freezing. RNA was extracted, DNAse treated and cDNA was made from 0.1 μg of RNA according to the SOP (PRCL-SOP-RNA purified cDNA synthesis). Gene expression levels were normalized to the endogenous housekeeping genes GAPDH and β-actin and relative expression was determined using the ΔΔCT method.

The highest levels of KCNV2-Opti expression were observed in retinal organoids transduced with AAV7m8-RK-codon-optimized KCNV2 compared to retinal organoids that received either AAV5-RK-codon-optimized KCNV2, AAV5-CAG-codon-optimized KCNV2, or AAV7m8-CAG-codon-optimized KCNV2 (Figure 15a).

The highest levels of vector-derived KCNV2 WT mRNA were observed in organoids transduced with AAV7m8-CAG-WT KCNV2. KCNV2 expression in organoids treated with AAV7m8-CAG-WT KCNV2 was approximately 138-fold higher than the non-transduced KCNV2 KO control (Figure 9b), and AAV7m8-RK-WT KCNV2 was approximately 86-fold higher than the non-transduced control. Retinal organoids transduced with different versions of the KCNV2 WT gene delivered with AAV5 under the control of either the CAG or RK promoter were approximately 10-fold higher than the non-transduced control (Figure 15B).

Overall, AAV2-7m8 was found to be more effective than AAV5 in transducing photoreceptors in retinal organoids. Interestingly, there were no significant differences in either WT KCNV2 mRNA or Opti KCNV2 mRNA in vectors driven by the photoreceptor-specific RK promoter or the constitutive CAG promoter.

Kv8.2 protein levels in transduced organoids assessed by immunofluorescence.
Kv8.2 protein levels were expressed in the outer nuclear layer of transduced organoids (see FIG. 7, FIG. 16B). To determine the relative cumulative protein levels between vectors, organoid cryosections were stained with Kv8.2 antibody and the total cumulative fluorescence (raw integrated density) of the ONL was quantified in FIJI (Image J) and normalized to the total ONL area measured. FIG. 16 shows the total fluorescence expressed as a percentage of WT organoids embedded in the same block and imaged on the same day. There was a significant difference in total fluorescence between the CAG and RK promoters in both 7m8 and AAV5 capsids (p=0.031 and 0.028, respectively, two-tailed, paired Student's t-test). Despite a trend for increased fluorescence intensity in the codon-optimized (Opti) vectors, there was no significant difference between WT and Opti in vectors containing the CAG or RK promoters.

Example 8: Colocalization of Kv8.2 and Kv2.1 in transduced retinal organoids.
Relative colocalization of Kv8.2 and Kv2.1 assessed by immunofluorescence Kv8.2 functions in the retina by forming a heteromer with the voltage-gated potassium channel Kv2.1. Qualitative analysis revealed that vector-derived Kv8.2 and endogenous Kv2.1 colocalized in the photoreceptor inner segment (Figure 12). Quantitative immunofluorescence and colocalization analysis was performed to determine the relative levels of restored Kv8.2. 7 um organoid cryosections were co-stained with Kv8.2 and Kv2.1, whole organoid sections were imaged at 40x magnification, and then merged in LSM software to generate tile scans of whole organoids, which were exported to FIJI image analysis software. Tile scans of n=3-5 organoids per vector were acquired and analyzed in FIJI (Image J). Kv. To determine pixels above threshold in both the Kv2.1 and Kv8.2 channels, the "image calculate>and" function was used to determine the total colocalization area of the inner segment region, which was normalized to the length of the region of interest to account for the various sizes of retinal organoids.

There was a significant difference between WT(CTR) and untreated KCNV2 KO organoids (Figure 16). There was a trend towards increased mean colocalization area in organoids treated with 7m8 CAG-WT and 7m8 CAG-Opti, but the differences did not reach significance (one-way ANOVA, Dunnett's multiple comparison test). The mean colocalization area of 7m8 RK-WT and RK-Opti was similar to untreated, indicating a lack of vector-derived Kv8.2 expression detectable by this method.

Colocalization and Proximity of Kv8.2 and Kv2.1 Assessed by Proximity Ligation Assay To assess protein-protein interactions in photoreceptors between potassium channel subunits Kv8.2 and Kv2.1, a proximity ligation assay (PLA) was developed. Transduced KCNV2 KO retinal organoids, together with WT (positive control) and non-transduced KO (negative control), were fixed and embedded in OCT on the same block for cryosectioning. 7 μm cryosections were co-stained with Kv8.2 (rabbit) and Kv2.1 (mouse) antibodies, and rabbit and mouse PLA plus and minus probes. After ligation and amplification steps (duo link-orange), PLA puncta in the outer nuclear layer were visualized by confocal microscopy at 63x magnification. When the organoids were examined as a whole, the PLA signal was clearly concentrated at the location of the photoreceptor cell layer, specifically where the photoreceptor inner segments were located (Fig. 18A). This confirms the specificity of the Kv.8.2/Kv2.1 interaction in the expected cell type and subcellular compartment. Furthermore, the specificity was confirmed by a significant reduction in the signal in the ONL of KCNV2 KO retinal organoids (Fig. 18B, 18C). Quantification of the PLA signal revealed a significant reduction in the number of PLA puncta per area of the ONL in KCNV2 KO clones (K28 and K12) compared to WT organoids treated on the same slide (Fig. 18C).

Quantification of PLA signal in transduced retinal organoids KCNV2 KO retinal organoids transduced with AAV vector express KCNV2 mRNA and Kv8.2 protein. The function of the translated protein derived from the vector depends on its ability to form heteromers with the voltage-dependent potassium channel kv2.1. In addition to assessing the total amount of Kv8.2 in the KCNV2 transcript and protein derived from the vector, PLA was used to assess the degree of interaction with Kv2.1.

The experiment was repeated with KCNV2 KO clonal cell lines K12 and K28, with KCNV2 KO organoids transduced with one of the eight indicated vectors from the clonal lines embedded in the same cryopreserved tissue blocks as WT (positive control) and non-transduced KCNV2 KO organoids (negative control). 7 μm cryosections were co-stained with Kv8.2 (rabbit) and Kv2.1 (mouse) antibodies and rabbit and mouse PLA plus and minus probes as described above. Maximum intensity z-projections at 63x magnification were used to quantitate PLA puncta per field. Each z-projection captured 100-150 photoreceptor cells and contained between 17 (non-transduced) and 550 puncta (maximum signal). The Kv8.2 antibody titer was reduced 100- to 400-fold to maximize signal without coalescence of PLA puncta. FIG. 19 shows representative maximum intensity projections from KCNV2 KO clone K12 transduced with both AAV2/5 and AAV7m8 capsids versus WT and non-transduced. PLA puncta were abundant in transduced compared to non-transduced organoids, with signal frequency highest in the apical region of the photoreceptor inner segment. Puncta in the ONL were quantified using the FIJI (Image J) "analyse particles" function. Regional differences in ONL size necessitated normalization of signal to the measured ONL area.

There was a significant effect of AAV transduction on PLA puncta counts (P>0.001, one-way ANOVA), with individual comparisons showing that all vectors produced significantly higher signals than non-transduced (Figures 20A and 20B). The CAG WT vector produced significantly higher PLA signals than the RK WT vector (K12 p=0.02, K28 p=0.01), but there was no significant difference between the CAG Opti and RK Opti vectors (K12 p=0.17, K28 p=0.77). In both clones, there was a significant difference between 7m8 RK Wt and 7m8 RK Opti (K12 p<0.0001, K28 p>0.01).

Higher PLA signal in KCNV2 KO photoreceptors is indicative of higher functional protein levels. In all 7m8 vectors, except for 7m8 RK-WT in clone K12, the signal was not significantly different from WT levels.

This indicates that all 7m8 vectors are capable of delivering KCNV2 to human photoreceptor cells with sufficient efficacy to allow restoration of functional Kv2.1/Kv8.2 heteromers to WT levels.

Claims (36)

(a) a promoter sequence that confers expression in photoreceptor cells, and (b) a nucleic acid sequence encoding Kv8.2;
wherein said nucleic acid sequence is operably linked to said promoter. The expression construct according to claim 1, wherein the promoter sequence is a CAG or rhodopsin kinase (RK) promoter sequence. The expression construct of claim 2, wherein the promoter sequence comprises a sequence that is at least 90% identical to SEQ ID NO:8. The expression construct of claim 3, wherein the promoter sequence comprises the sequence of SEQ ID NO:8. The expression construct of claim 2, wherein the promoter sequence comprises a sequence that is at least 90% identical to SEQ ID NO:7. The expression construct of claim 5, wherein the promoter sequence comprises the sequence of SEQ ID NO:7. The expression construct according to any one of claims 1 to 6, further comprising a post-transcriptional regulatory element. The expression construct of claim 7, further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The expression construct of claim 7, wherein the WPRE comprises a sequence that is at least 90% identical to SEQ ID NO:11. The expression construct of claim 9, wherein the WPRE comprises a sequence comprising SEQ ID NO:11. The expression construct according to any one of claims 1 to 10, wherein the nucleic acid sequence encoding Kv8.2 is a WT KCNV2 gene. The expression construct according to any one of claims 1 to 10, wherein the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 90% identical to SEQ ID NO:9. The expression construct of claim 12, wherein the nucleic acid sequence encoding Kv8.2 comprises a sequence comprising SEQ ID NO:9. The expression construct according to any one of claims 1 to 10, wherein the nucleic acid sequence encoding Kv8.2 is a codon-optimized KCNV2 gene sequence. The expression construct according to any one of claims 1 to 10, wherein the nucleic acid sequence encoding Kv8.2 comprises a sequence that is at least 90% identical to SEQ ID NO: 10. The expression construct of claim 15, wherein the nucleic acid sequence encoding Kv8.2 comprises a sequence comprising SEQ ID NO: 10. The expression construct according to any one of claims 1 to 16, wherein the nucleic acid sequence encoding Kv8.2 encodes a protein comprising a sequence that is at least 90% identical to SEQ ID NO: 13. The expression construct of claim 17, wherein the nucleic acid sequence encoding Kv8.2 encodes a protein comprising SEQ ID NO: 13. The expression construct according to any one of claims 1 to 18, wherein the expression construct further comprises a bovine growth hormone polyadenylation (BGH-polyA) signal. 20. The expression construct of claim 19, wherein the polyadenylation signal comprises a sequence that is at least 90% identical to SEQ ID NO:12. The expression construct of claim 20, wherein the polyadenylation signal comprises SEQ ID NO: 12. The expression construct according to any one of claims 1 to 21, comprising a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4. The expression construct according to claim 22, comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 4. A vector comprising an expression construct according to any one of claims 1 to 23. The vector according to claim 24, which is a viral vector. The vector according to claim 25, which is an adeno-associated virus (AAV) vector. The vector according to claim 26, comprising a genome derived from AAV serotype AAV2. The vector according to any one of claims 26 or 27, comprising a capsid derived from AAV7m8. The vector according to any one of claims 26 or 27, comprising a capsid derived from AAV5. A pharmaceutical composition comprising the vector according to any one of claims 24 to 29 and a pharma- ceutical acceptable carrier. A method for treating a retinal disease in a subject in need of such treatment, wherein the retinal disease is associated with one or more mutations in the KCNV2 gene, the method comprising administering to the subject a vector according to any one of claims 24 to 29 or a pharmaceutical composition according to claim 30. The method of claim 31, wherein the retinal disease is cone dystrophy with supernormal rod response (CDSSR). A method for increasing KCNV2 expression in a subject in need of increased expression of KCNV2, comprising administering to the subject a vector according to any one of claims 24 to 29 or a pharmaceutical composition according to claim 30. A method for increasing Kv8.2 levels in photoreceptors in a subject in need of increased Kv8.2 levels, comprising administering to the subject a vector according to any one of claims 24 to 29 or a pharmaceutical composition according to claim 30. The method according to any one of claims 31 to 34, wherein the vector or pharmaceutical composition is administered by intraocular injection. 36. The method of claim 35, wherein the vector or pharmaceutical composition is injected into the central retina of the subject.